Osteoporosis is a disease that disorder of osteoclasts and osteoblasts results in reducing bone densities and changing bone structures and further results in increasing the ratio of fracture. Osteoporosis companying with occurring of fracture does not only cause pain to human bodies but also increases patients' death ratio and serious economic burdens.
Osteoblasts, adipocytes, and fibroblasts are all divided from mesenchymal stem cells. However, osteoclasts are divided from macrophage/monocyte linage of hematopoietic cells. The two cytokinins, macrophage colony-stimulating factor, M-CSF, and receptor activator of nuclear factor-κB ligand, RANKL, which are secreted by bone marrow stromal cells and Osteoblasts and belong to tumor necrosis factor, TNF, are the essential factors for monocytic progenitor cells dividing to osteoclasts. RANKL is expressed on the surfaces of osteoblasts, bone marrow stromal cells and vascular endothelial cells. Moreover, RANKL can combine with RANK of monocytic progenitor cells' surface and stimulate cells to divide into coenocytic osteoclasts. At this time, maturely-divided osteoclasts secrete tartrate-resistant acid phosphatase, TRAP, to perform an ability of dissolving bones.
RANKL/RANK
RANK, the receptor of RANKL, is usually expressed on the cell membranes of progenitor cells of osteoclasts and maturely-divided osteoclasts. RANKL directly interacts with RANK by contact between cells, and stimulates osteoclasts to differentiate and proliferate to perform an ability of dissolving bones. After RANKL combines with RANK, RANK will interact with tumor necrosis factor receptor-associated factor, TRAFs, and switches on series signal transduction pathways of differentiation and activation of osteoclasts which include three major signal transduction pathways: (1) nuclear factor Kappa B, NF-κB, (2) mitogen activated protein kinase, MAP kinase or MAPK, and (3) Src/PI3K/Akt signal transduction pathways and important transcription factors, c-Fos and NFATc 1. NF-κB and MAP kinase signal transduction pathways regulate the ability of differentiation and dissolution of the osteoclasts. Also Wong, B R. etc. 1999, Mol. Cell. Vol. 4(6) 1041-1049 hold that Src/PI3K/Akt signal transduction pathway is the pathway that dominates survival of osteoclasts.
TRAFs
TRAF family includes six different proteins which can interact with RANK. Misukami, J. etc. 2002. Mol. Cell. Biol. Vol. 22(4) 992-1000 discovered that the mice only knocked down TRAF6 gene would have osteopetrosis due to severe function defect of osteoclasts. Moreover, Armstrong, A P. etc. 2002. J. Biol. Chem. Vol. 277(46) 44347-44356 discovered that mutating mouse gene, RANK, to make RANK lack binding sites of TFAF6 and let monocytes lose the ability of dividing to osteoclasts. It shows TRAF6 plays an important role in NF-κB activating RANKL/RANK signal transduction pathway.
Nuclear Factor κB, NF-κB
NF-κB will combine with inhibitory κB and maintains inactive condition under non-stimulating condition. When NF-κB is activated by RANKL, RANKL will combine with RANK and stimulate NF-κB to activate through TRAF6. The activation of NF-κB lead to phosphorylation on IκB at Ser-32 and Ser-36 by IκB kinase, IKK, and then the NF-κB proceeds hydrosis to separate from IκB. After that, the NF-κB will enter an nucleus to induce many genes relating to a dissolution activity of osteoclasts.
Mitogen-Activated Protein Kinases, MAP Kinase, MAPK, Pathway
The members of mitogen-activated protein kinases, MAP kinase or MAPK, family include extracellular signal-regulated kinase, ERK, p38-MAPKs, and c-Jun N-terminal kinase, JNK. It is known that p38's inhibitor, SB203580, or ERK's inhibitor, PD98059, both can inhibit induction of osteoclasts' differentiation and osteoclastogenesis by RANKL. The activation of ERK and JNK will affect the function of downstream transcription factor, activator protein-1, AP-1. ERK can induce and active the expression of c-Fos protein which is one of AP-1 family. Moreover, JNK can control the production of osteoclasts by phosphorylating c-Jun and enhancing transcription activities of Ap-1.
Src/PI3K/Akt Pathway
Src interacting with TRAF6 plays an important role in RANKL activating anti-apoptotic serine/threonine kinase, Akt. Src binds on the tail of RANL of cytoplasm and is activated by the stimulation that RANKL binds on RANK and TRAF6. Then, Src activates down stream, Akt. Moreover, in the process that Src activates Akt, the medium of phosphatidylinositol 3-kinase, PI3K, is needed. Using Src inhibitor, removing Sre gene or using LY294002 which is the inhibitor of PI3K can inhibit the activation of Akt induced by RANKL. RANKL/RANK regulates the surviving ability of osteoclasts through TRAF6 via the signal pathway of Src/PI3K/Akt.
Transcription Factors
RANKL/RANK signal pathway stimulates many osteoclast dissolution related gene expressions via activating transcription factor, AP-1, and NF-κB. In Ap-1 family, c-Jun, JunB, c-Fos, Fra-1 and Fra-2 participate in the regulation of generating osteoclasts. Furthermore, after c-Fos interacts with transcription factor such as nuclear factor of activated T cells, NFATc1, it stimulates the expression of the osteoclast dissolution genes, TRAP and Cathepsin K. Takayanagi, H. et al in 2007, Ann. NY Acad. Sci. No. 1116, Page 227-237: By calcium/calmodulin signal transduction of the up stream, after inner cellular calcium binds to calcium binding protein, calmodulin, first, it activates calcium/calmodulin-activated kinases, CaMKs and calcinerurin to make NFATc1 active. Therefore, Ap-1, NFATc1 and NF-κB are key transcription factors of regulating osteoclast dissolution.
Pro-Inflammatory Cytokines
Such as TNF-α, interleukin-1 (IL-1) and IL-6 can promote inflammation to induce the generation of osteoclasts. TNF-α which is released by active T cells and macrophages can promote the expression of RANK and increase the sensitivity of RANK for RANKL. Under inflammation, IL-6, IL-11 and IL-17 promote bone loss. However. Wei, S. et al in 2008, Pathol. Res. Pract. Section 204 (10), pages 695-706 found that IL-4, IL-10, IL-12, IL-18 and interferon-yby inhibiting osteoclast differentiation and the dissolution activity thereof to inhibit the formation of osteoclasts in vivo and in vitro.
High-Mobility Group Box 1, HMGB1
HMGB 1 is a protein in Eukaryotic cell nucleus. When macrophages are activated or influenced by pro-inflammatory cytokines, HMGB 1 will transfer to cytoplasm from nucleus, and then it will be moved out of cell by exocytosis or cell membrane break. Zhou, Z. et al in 2008, J. Bone. Miner. Res. Vol. 23(7), page 1084-1096 found that extracellular HMGB1 is similar to pro-inflammatory cytokines and promotes the bone loss under inflammatory conditions.
Monosodium Iodoacetate, MIA
Monosodium iodoacetate, MIA, is the glyceraldehyde-3-phosphate dehydrogenase, GAPDH, inhibitor, and in vitro, it can inhibit glycolysis to cause death of chondrocyte cells. In vivo, to inject MIA into knee joint cavities can induce the death of chondrocyte cells of knee joints to cause osteoarthritis. Under inflammatory environments, lymphocytes, macrophages and mast cells are infiltrated, and these cells release pro-inflammatory cytokines to activate osteoclasts, and then it promotes the bone loss of articular cartilage and subchondral bone.
With theophylline structure, KMUP-1 which is modified by the chlorophenyl is with the chemical name, 7-[2-[4-(2-Chlorophenyl) piperazin-1-yl]ethyl] theophylline. The theophylline structure is as Formulation 1, and the structure of KMUP-1 is as Formulation 2.

It is known that KMUP-1 has abilities of activating eNOS of epitheliums and endothelium, partial activating smooth muscle soluble guanylyl cyclase, sGC, inhibiting 3′, 4′ and 5′-phosphodiesterase, PDE, to increase on the amount of intercellular cGMP and opening potassium channels. Furthermore, KMUP-1 can induce the caversonal smooth muscle of rabbits' penises to be relaxed via stimulating the mechanism of cGMP and opening potassium channels. Besides, KMUP-1 has been proved that it can influence cAMP/PKA and cGMP/PKG pathway to cause the increase of the amount of tracheal epithelium's NO and to further activate the sGC in tracheal smooth muscle cells. Or KMUP-1 directly activates the sGC of smooth muscle cells to increase the amount of cGMP and activate PKG. KMUP-1 can also directly activate adenylate cyclase to induce the increase in the amount of cAMP to activate PKA. PKA and PKG both can cause the opening of potassium channels of smooth muscle cell membrane to make tracheal smooth muscle relaxed eventually. cAMP and cGMP are second transduction factors and can also control many physiological responses which include cell growth, cell differentiation, apoptosis, glycolysis, ester hydrolysis, immunization and inflammation,
Miyamoto, K. et al in 1997, Biochem. Pharmacol. 54 (5), Page 613-617 found that in Walker256/S-bearing rat model, PDE4 inhibitors can inhibit the bone loss; Yoshimura, T. et al in 1997, Gen. Pharmacol. Vol. 29 (4), Page 633-638 are also considered that the proliferation of the key cytokines, TNF-α, IL-1 and IL-6, of bone loss can be inhibited by PDE4.